Detecting multinucleotide repeats

Abstract

Methods of determining the length of a multinucleotide repeat region in a target nucleic acid are provided herein which include labeling amplified target nucleic acids with a target detection label independent of the number of multinucleotide repeats and a repeat-detection label proportional to the number of multinucleotide repeats, wherein the two types of labels are each independently incorporated in the amplified target nucleic acids during the amplifying or after the amplifying; binding the amplified target nucleic acids to a capture probe specific for the amplified target nucleic acids; detecting the target detection label associated with the capture probe to produce a first signal; detecting the repeat-detection label associated with the capture probe to produce a second signal; and determining a ratio of the first signal and the second signal, wherein the ratio is indicative of the length of the multinucletotide repeat region in the target nucleic acid.

1 . A method of determining the length of a multinucleotide repeat region in a target nucleic acid, comprising:
 amplifying a target nucleic acid comprising a multinucleotide repeat region to produce amplified target nucleic acids incorporating a straddle primer labeled with a first label, amplified target nucleic acids thereby labeled with the first label independent of the number of multinucleotide repeats;   labeling the amplified target nucleic acids with a second label, the second label proportional to the number of multinucleotide repeats, wherein the second label is incorporated in the amplified target nucleic acids during the amplifying or after the amplifying, wherein the first and second labels are different;   binding the amplified target nucleic acids to a capture probe specific for the amplified target nucleic acids;   detecting the first label associated with the capture probe to produce a first signal;   detecting the second label associated with the capture probe to produce a second signal; and   determining a ratio of the first signal and the second signal, wherein the ratio is indicative of the length of the multinucleotide repeat region in the target nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein binding of the amplified target nucleic acids comprises specific hybridization of the amplified target nucleic acids to complementary nucleic acid capture probes. 
     
     
         3 . The method of  claim 1 , wherein the second label is present in nucleotides used in amplifying the target nucleic acid to produce the amplified target nucleic acids. 
     
     
         4 . The method of  claim 1 , wherein the second label is present in probes which specifically bind to multinucleotide repeats in the amplified target nucleic acids. 
     
     
         5 . The method of  claim 4 , wherein the probes are nucleic acid probes. 
     
     
         6 . The method of  claim 1 , wherein the capture probe is specific for the multinucleotide repeat region of the amplified target nucleic acids. 
     
     
         7 . The method of  claim 1 , wherein the capture probe comprises an encoded substrate. 
     
     
         8 . The method of  claim 1 , wherein the target nucleic acid is isolated from a biological sample. 
     
     
         9 . The method of  claim 1 , wherein the target nucleic acid is genomic DNA. 
     
     
         10 . The method of  claim 8 , wherein the biological sample is obtained from an individual subject. 
     
     
         11 . The method of  claim 9 , wherein the individual subject is human. 
     
     
         12 . The method of  claim 10 , wherein the individual subject has or is at risk of having a trinucleotide repeat expansion disorder selected from the group consisting of: Dentatorubropallidoluysian atrophy, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type 3, spinocerebellar ataxia type 6, spinocerebellar ataxia type 7, spinocerebellar ataxia type 17, fragile X syndrome; fragile XE mental retardation; Friedreich's ataxia; myotonic dystrophy; spinocerebellar ataxia type 8 and spinocerebellar ataxia type 12. 
     
     
         13 . The method of  claim 7 , wherein the encoded substrate is a plurality of encoded particles, producing a first particle set. 
     
     
         14 . The method of  claim 1 , further comprising:
 amplifying a reference nucleic acid multinucleotide repeat region to produce amplified target reference nucleic acids;   labeling the amplified target reference nucleic acids with a first label and a second label, the first label independent of the number of multinucleotide repeats and the second label proportional to the number of multinucleotide repeats, wherein the first and second labels are each independently incorporated in the amplified target reference nucleic acids during the amplifying or after the amplifying;   binding the amplified target reference nucleic acids to a capture probe specific for the amplified target reference nucleic acids;   detecting the first label associated with the capture probe to produce a third signal;   detecting the second label associated with the capture probe to produce a fourth signal;   determining a ratio of the third signal and the fourth signal, wherein the ratio is indicative of the length of the multinucleotide repeat region in the target reference nucleic acid; and   comparing ratio of the first signal and the second signal with the ratio of the third signal and the fourth signal.   
     
     
         15 . The method of  claim 1 , further comprising:
 amplifying a second target nucleic acid to produce amplified second target nucleic acids;   labeling the amplified second target nucleic acids with a first label and a second label, the first label independent of the number of multinucleotide repeats and the second label proportional to the number of multinucleotide repeats;   binding the amplified second target nucleic acids to a capture probe specific for the amplified second target nucleic acids;   detecting the first label associated with the capture probe to produce a first signal;   detecting the second label associated with the capture probe to produce a second signal; and   determining a ratio of the first signal and the second signal, wherein the ratio is indicative of the length of the multinucleotide repeat in the second target nucleic acid.   
     
     
         16 . The method of  claim 15 , wherein the second encoded substrate is a plurality of encoded particles, producing a second particle set. 
     
     
         17 . The method of  claim 16 , wherein the first and second particle sets are present together in a reaction vessel during binding of the amplified first and second target nucleic acids to the first and second encoded substrates. 
     
     
         18 . A method of screening an individual for a genetic condition characterized by an altered multinucleotide repeat region in a target nucleic acid, comprising:
 amplifying from a sample obtained from the individual a target nucleic acid to produce amplified target nucleic acids, wherein the amplified target nucleic acids contain a first label, the first label independent of the number of multinucleotide repeats and the second label proportional to the number of multinucleotide repeats;   binding the amplified target nucleic acids to a capture probe specific for the multinucleotide repeat of the amplified target nucleic acids;   detecting the first label associated with the capture probe to produce a first signal;   detecting the second label associated with the capture probe to produce a second signal;   determining a ratio of the first signal to the second signal, wherein the ratio is indicative of the length of the multinucleotide repeat in the target nucleic acid, and   comparing the determined ratio with that from a control sample to determine the presence of an altered multinucleotide repeat region in the individual.   
     
     
         19 . A composition, comprising:
 amplified target nucleic acids comprising a multinucleotide repeat region, the amplified target nucleic acids comprising a first label and a second label, the first label independent of the number of multinucleotide repeats and the second label proportional to the number of multinucleotide repeats, wherein the first and second labels are each independently incorporated in the amplified target nucleic acids during the amplifying or after the amplifying, wherein the amplified target nucleic acids are bound to a capture probe specific for the amplified target nucleic acids.